Mutagenesis vol. 19 no. 1 pp. 75-80,
January 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
In vitro genotoxicity testing of the furylethylene derivative UC-245 in human cells
1Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain and 2Centro de Bioactivos Químicos, Universidad Central de Las Villas, Santa Clara, Cuba
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Abstract |
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The possible genotoxic potential of the 2-furylethylene derivative UC-245 has been evaluated in vitro using human cells as a test system. This compound was synthesized at the Centro de Bioactivos Químicos, Universidad Central de Las Villas (Cuba) and it appears to be effective against leishmaniosis. The induced genetic damage was determined by scoring the frequency of micronuclei (MN) and the frequency of sister chromatid exchanges (SCE) in primary lymphocyte cultures set up from two different donors. The DNA breakage level was also evaluated by the Comet assay, using an established human lymphoblastoid cell line (TK6). For the MN and SCE studies, to detect eventual metabolic modification in the genotoxicity of this compound, the cultures were treated with S9 microsomal fraction. The results obtained indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of presence/absence of the metabolic fraction, which would indicate a lack of clastogenic and/or aneugenic potential. Nevertheless, a clear and significant increase in the SCE frequency was observed in the treatments without S9. This would support the 2-furylethylene derivative UC-245 inducing DNA primary damage. In addition, the results obtained in the Comet assay also show that UC-245 induces a significant increase in the level of DNA breakage, which would confirm its genotoxicity.
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Introduction |
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The compound UC-245 is one of the members of a family of furylethylene derivatives synthesized in the laboratories of the Centro de Bioactivos Químicos, Universidad Central de Las Villas (Cuba). Interest in the study of these compounds has increased in recent years due to the potent microcidal activity shown by compounds with this type of chemical structure (Estrada, 1998
).
At present, compound UC-245 seems to be effective against leishmaniosis, as shown in a murine model (Monzote et al., 1999). This is a complex pathology that affects both man and animals, caused by different flagellate protozoa belonging to the genus Leishmania. It is estimated that the prevalence of this illness is 
12 000 000 people distributed around the world, although mostly in Central and South America. Due to the low efficiency of the commonly used drugs, the World Health Organization (2000) has stimulated research on new drugs for the treatment of this illness.
All new drugs, in addition to their new therapeutic advantages, must show low or null side effects. In this context, knowledge of the potential genotoxic risk associated with any particular compound is of paramount importance. Our group has been studying the genotoxicity of different 2-furylethylene derivatives (González Borroto et al., 2001, 2002). In this novel group of derivatives, the nitro group is not attached to the furan ring, but is placed at the double bond present in the ethylenic chain at the ß-carbon atom. Several studies have demonstrated that only the 5-nitrofurans are mutagenic and that derivatives with the nitro group outside the furan ring do not show mutagenic activity (Sturdick et al., 1985, 1986).
To determine the genotoxic potential of compound UC-245, three well-known genotoxicity assays were performed: the in vitro micronucleus (MN) assay using the cytokinesis block technique, the sister chromatid exchanges (SCE) assay and the single cell gel electrophoresis assay, also called the Comet test. The use of different assays measuring different genetic end points gives us a clearer picture of the real genotoxicity profile of the test compound in mammalian cell systems. It must be remembered that the MN assay measures both the clastogenic and aneugenic potential, the SCE assay measures primary DNA damage, whilst the Comet assay mainly shows DNA breakage.
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Materials and methods |
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Cells used
The study was carried out using both human peripheral lymphocytes from fresh blood samples and an established lymphoblastoid cell line (TK6). Blood was obtained from two healthy, non-smoking men, aged 25 and 35 years old. Approximately 20 ml of blood was collected, by venipuncture, into syringes containing sodium heparin as anticoagulant. Lymphocyte cultures were used for the MN and SCE assays. The TK6 cell line was used for the Comet test.
Test chemicals
The test chemical UC-245 [1-(5-bromofur-2-yl)-2-methyl 2-nitroethene] used in this study was synthesized at the Centro de Bioactivos Químicos (CBQ, Villa Clara, Cuba). A particular characteristic of this compound is that it has the nitro group located in the exocyclic double bond position C2 (Figure 1). Preliminary toxicity experiments were conducted to determine the dose ranges used in the genotoxicity assays. A range of concentrations of the test agent (from 2.5 to 20 µg/ml) was used for the MN assay. For the SCE assay the range of doses used was from 1.25 to 20 µg/ml. The range for the Comet assay was from 1.25 to 30 µg/ml. These ranges were selected by taking into account the survival and the cytotoxic/cytostatic effects found in previous dose range studies. The test agent was dissolved in dimethyl sulfoxide (DMSO) (CAS no. 67-68-5; Panreac, Barcelona, Spain) at a final volume in culture of 1% of the total. DMSO was also tested as a solvent control.
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In the cultures without metabolic activation, the positive control for testing MN was mitomycin C (MMC) (CAS no. 50-07-7; Sigma Chemical Co., St Louis, MO), at 2 and 0.2 µM for treatments lasting 3 and 48 h, respectively. For cultures with metabolic activation, cyclophosphamide (CP) (CAS no. 6055-19-2; Sigma) at 0.1 mM was used. For SCE demonstration without metabolic activation, MMC at 0.5 and 0.1 µM was used for treatments lasting 3 and 48 h, respectively, whilst in cultures with metabolic activation, 0.005 µM CP was used. The positive control used in the Comet assay was 150 µM H2O2 (CAS no. 7722-84-1; Sigma) and the treatments lasted for 3 h.
Metabolic activation
The metabolic activation fraction used was S9 from male Sprague–Dawley rats induced with Aroclor 1254 (ICN Biomedicals Inc., Aurora, OH). The freshly prepared S9 mix consisted of 1 ml of S9, 0.33 ml of 1 M KCl, 0.32 ml of 0.25 M MgCl2·6H2O, 0.25 ml of 0.2 M glucose 6-phosphate, 1 ml of 0.04 M NADP, 2.10 ml of distilled water and 5 ml of phosphate buffer (pH 7.4).
Lymphocyte cultures: MN and SCE assays
Two blood cultures were established for each genetic end point and concentration. A whole blood sample (0.5 ml) was added to 4.5 ml of culture medium composed of 3.69 ml (82%) RPMI 1640 medium supplemented with 0.675 ml (15%) heat-inactivated fetal calf serum, 0.045 ml (1%) phytohemagglutinin (PHA), 0.045 ml (1%) L-glutamine and 0.045 ml (1%) antibiotics (5000 IU penicillin and 5000 µg/ml streptomycin). All compounds were obtained from Gibco (Barcelona, Spain).
The cytokinesis block micronucleus assay (CBNM) was carried out using the standard technique proposed by Fenech (1993), with slight modifications following the methodology established in our laboratory and detailed in previous works (Surrallés et al., 1992, 1994). The experiments for SCE demonstration were performed similarly, as recently reported (González Borroto et al., 2001). Staining of SCE was conducted following the fluorescence plus Giemsa method (Perry and Wolff, 1974).
The procedures indicated above were used for the assays conducted with and without metabolic activation. Nevertheless, in the case of cultures with metabolic activation, 24 h after the initiation of cultures 0.5 ml of S9 mix was added together with the test agent. After an incubation period of 3 h at 37°C, the test chemical and S9 mix were removed from the culture. Concurrent cultures, treated for 3 h without the activating system, were also set up. The lymphocyte pellet was washed twice with 5 ml of RPMI 1640 medium, resuspended in complete medium and finally the cultures were incubated for 72 h at 37°C.
Microscopic observation
All slides were coded prior to scoring. MN and SCE scoring was done by the same person (G.P.M.) using a Leitz-Leica light microscope at 1000x magnification under oil immersion. The criteria for MN scoring were as described by Fenech (1993) and recently reviewed by Kirsch-Volders et al. (2000).
Five hundred cells were scored to determine the percentage of cells with one to four nuclei. The number of MN in 1000 binucleated (BN) cells was scored for each treatment and the number of BN cells with MN were also recorded. A cytokinesis block proliferation index (CBPI) was calculated according to the formula:
CBPI = [MI + 2MII + 3(MIII + MIV)]/N
where MI–MIV represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored (Surrallés et al., 1995).
For the SCE analysis, 50 second division metaphases were examined for each treatment and donor. A further 100 metaphases for each concentration were also scored to evaluate the proportion of cells that had undergone one, two or three divisions. The proliferative rate index (PRI) was calculated following the expression:
PRI = (M1 + 2M2 + 3M3)/N
where M1, M2 and M3 indicate those metaphases corresponding to the first, second and third divisions and N is the total number of metaphases scored (Ribas et al., 1998).
Comet assay
The Comet assay was performed as previously described (Singh et al., 1988), with some modifications. The cell samples (40 000 cells, 20 µl) were carefully resuspended in 75 µl of 0.5% low melting point agarose (LMA), layered onto microscope slides precoated with 150 µl of 0.5% normal melting point agarose (dried for 10 min at 65°C) and spread with a coverslip. After solidification for 10 min at 4°C the coverslips were removed, another 75 µl of 0.5% LMA was placed on the slides, covered with a coverslip and kept for 15–20 min at 4°C. The slides were then immersed without the coverslips in cold fresh lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100 and 1% laurosylsarcosinate, pH 10) for 2 h at 4°C in a dark chamber. To prevent the occurrence of additional DNA damage, the following steps were performed under dim light. The slides were placed for 40 min in a horizontal gel electrophoresis tank filled with cold electrophoresis buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13.5) to allow DNA unwinding. Electrophoresis was performed in the same buffer for 20 min at 0.73 V/cm and 300 mA. Unwinding and electrophoresis treatments were made in an ice bath. After electrophoresis, the slides were neutralized twice for 5 min with 0.4 M Tris (pH 7.5) and fixed with 3 ml of absolute ethanol for 3 min. The slides were stained with 50 µl of ethidium bromide (0.4 µg/ml) just before analysis. Finally, the images were examined at 400x magnification with a Komet 3.1 Image Analysis System (Kinetic Imaging Ltd, Liverpool, UK), fitted with an Olympus B x 50 fluorescence microscope equipped with a wide band excitation filter of 480–550 nm and a barrier filter of 590 nm. Two parallel replicate slides were performed per sample, scoring a total of 100 randomly selected cells per sample. The Olive tail moment was computed as a measure of DNA damage.
Statistical analysis
Analysis of the BN cells with MN was performed for each treatment using the one-tailed Fisher’s exact test. The 
2 test was used for the analysis of CBPI and PRI among the treatments. For the statistical analysis of SCE, we used the parametric t-test for independent samples. The normality of the distribution of SCE scores was assessed by means of the Kolmogorov–Smirnov test of goodness of fit and the equality of variances was verified with Levene’s test. Analysis of the Comet data was performed using SAS/PC v.8.0 (SAS Institute, Cary, NC). Statistical decisions were made at a significance level of 0.05.
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Results |
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The data obtained in this study measuring the induction of MN in cultured human lymphocytes using cytochalasin B, with and without S9 mix, are summarized in Tables I–III. Table I shows that a treatment lasting 48 h does not significantly increase the frequency of binucleated cells with MN. The slight positive response obtained in donor A at a concentration of 10 µg/ml can be considered marginal, based on the negative results found in donor B. The results obtained in the treatments lasting for 3 h, without and with metabolic activation, were also negative (Tables II and III), since no induction of MN was observed under any of the assay conditions.
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In spite of the lack of genotoxic activity found in the MN assay, cytotoxic/cytostatic effects of the test compound on lymphocyte cultures were observed and the CBPI values decreased with increasing doses of UC-245. The cytotoxic/cytostatic effects were mainly observed in donor B, at the highest concentration tested (10 µg/ml) and in the treatment lasting for 48 h, where no cell division was observed in the culture. Similarly, the concentration of 20 µg/ml was completely toxic and impossible to evaluate in the cultures treated for 3 h in absence of S9 fraction. Since this was not observed in the treatments with S9, it could indicate a possible mechanism of detoxification or the possibility that the enzymes in the S9 fraction deactivate the furylethylene derivative, leading to the formation of a metabolite with lower cytotoxicity.
Tables IV and V summarize the results obtained for the SCE assay. The frequencies of SCE obtained for both 3 and 48 h treatments indicate that compound UC-245 induces cytogenetic damage that results in an increase in SCE frequency. In both donors a clear, direct dose–response relationship was observed for the treatments without metabolic activation. For treatments with metabolic activation lasting 3 h a clear decrease in the response was found, although with large differences between donors. High variability between donors is often obtained, which could indicate the existence of different genetic polymorphisms related to metabolism. Our results confirm that the enzymes present in the S9 fraction transformed compound UC-245 to metabolites less effective in producing SCE.
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As found in the MN experiments, in the assays performed to demonstrate SCE the test compound showed clear cytotoxic/cytostatic effects when measured as cell cycle delay (PRI), and a reduction in cell proliferation was observed in both donors. Due to the high toxicity of the 10 and 20 µg/ml concentrations in donor A and for the treatment lasting 48 h, we were unable to evaluate the PRI for such levels of exposure.
The results obtained in the Comet assay after treatment of TK6 cells with six concentrations (from 1.25 to 30 µg/ml) of the test compound are presented in Figure 2. This figure shows the results obtained in three independent experiments, with treatments lasting for 30 min. In one experiment only four concentrations (1.25, 2.5, 5 and 10 µg/ml) were tested. We report here data corresponding to the Olive tail moment, where a clear positive dose-dependent relationship was observed. As expected, the positive control (H2O2) induced a significant response. In the Comet assay TK6 cells were used instead of human lymphocytes to avoid the high variability existing between cells within donors, in addition to inter-donor variability (Morillas et al., 2002). The treatments were only carried out without S9, to confirm the genotoxicity observed in the SCE assay, where the response was higher in the experiments carried out without metabolic activation.
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Discussion |
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Nitrofurans constitute an important group of chemicals with antimicrobial properties that are currently used in human and veterinary medicine. Although some of these compounds have been shown to exert genotoxic activity, many aspects of the genotoxic mechanisms of nitrofuran derivatives still remain unclear (Jurado and Pueyo, 1995
).
It has been indicated that the position of the nitro group is an important factor in modulation of the genotoxicity of nitrofurans. Thus, Sturdik et al. (1985, 1986), when studying the structure–mutagenicity relationship of 5-nitro-2-furylethylenes in Salmonella typhimurium, concluded that only those derivatives with the nitro group in position 5 of the furanic ring were mutagenic. This would agree with data on the electronic molecular structure of different 2-furylethylene derivatives reported by Estrada (1998) in a molecular orbital study, where marked electronic differences were found among compounds with the nitro group at different positions. Thus, when the nitro group is in the 5 position of the furan ring it is more sensitive to reduction in cell culture than when it is placed at position ß of the exocyclic double bond. In this context, it must be remembered that the compound UC-245 tested in this study has the nitro group located in the exocyclic double bond at position C2.
The results obtained with this compound indicated that, although UC-245 was unable to increase the frequency of MN, it showed clear genotoxic effects when evaluated in the SCE and Comet assays. These apparent discrepancies in the data suggest that the interaction of UC-245 with DNA does not induce any of the modifications detected by the MN test. It should be noted that the MN assay detects both clastogenic and aneugenic effects. The negative results obtained in the MN assay with UC-245 agree with those previously obtained with the compound G0, which is the structural precursor of UC-245 (González Borroto et al., 2001).
The positive response obtained in the SCE assay would indicate a clear genotoxic potential of UC-245, in spite of the negative response in the MN assay. This fact would support the need for a test battery in genotoxicity testing to avoid false negative data, since each individual test measures a different type of genetic damage. Thus, while the MN assay is especially sensitive to those agents inducing single- and/or double-strand breaks, the SCE assay is sensitive to those agents that bind covalently to DNA.
Several studies have demonstrated that a great number of mutagens increase the SCE frequency at far lower concentrations than those required to produce chromosome breakage. Thus, SCE analysis has been considered to be highly sensitive for measuring the mutagenic and carcinogenic potential of many environmental agents (Tucker et al., 1993). Our results would indicate that compound UC-245 is able to induce DNA damage that interferes with the replication mechanisms, leading to significant increases in the frequency of SCE. It is known that both DNA adducts and double-strand breaks, when repaired by recombinational mechanisms, are involved in the formation of SCE (Dong and Fasullo, 2003). Due to the negative effects of UC-245 in the MN assay, if SCE denote recombinational repair of DNA breaks, they should be of apoptotic origin rather than of direct genotoxic origin. Nevertheless, the positive results obtained in the Comet assay were obtained at concentrations inducing very low toxicity and no significant increases in comets without a head, indicative of apoptotic DNA cleavage, were observed.
Thus, the genotoxicity of UC-245 is confirmed by the results obtained in the Comet test. Due to its sensitivity for measuring DNA damage at the individual cell level and its potential application to virtually all eukaryotic cell types, the Comet assay has been adopted as a very useful short-term test in genotoxicity studies (McKelvey-Martin et al., 1993).
Both the SCE and Comet results reveal the genotoxicity of compound UC-245, which does not agree with the assumption that furylethylene derivatives with the nitro group at position ß of the exocyclic double bond have a low genotoxic potential.
There is experimental evidence indicating a possible detoxification pathway for nitrofurans and furylethylenes. Studies carried out by Ramos et al. (1997) on the mutagenicity of a substituted nitroalkene 1-(5-bromofur-2-il)-2-bromo-2-nitroethene in the Salmonella/microsome assay reported that genotoxicity was reduced when the S9 metabolic activation fraction was used. In addition, in the mouse bone marrow micronucleus test, they did not find genotoxicity, which may suggest the involvement of a detoxification process. Similar results have also been obtained by us when testing the compound G1, where presence of the S9 fraction reduced the genotoxicity observed in the SCE assay (González et al., 2002), and no genotoxicity was observed in the mouse bone marrow micronucleus test (González Borroto et al., 2003). The results from the present study would agree with the previous data, since the effects observed in the SCE assay are reduced when S9 mix is added to the culture. Taking into account the results obtained with metabolic activation, we consider that in vivo studies using the tests reported here are needed to obtain further information for risk assessment.
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Acknowledgements |
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We would like to thank G. Umbert for her expert technical help. G. Pérez Machado was supported by a fellowship from the Spanish Agency of International Cooperation (AECI). This investigation was supported in part by the Generalitat de Catalunya (2002SGR-00197).
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Notes |
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3To whom correspondence should be addressed. Fax: + 34 93 5812387; Email: ricard.marcos{at}uab.es
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