Mutagenesis Advance Access originally published online on April 7, 2005
Mutagenesis 2005 20(3):193-197; doi:10.1093/mutage/gei026
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Comparative genotoxic evaluation of 2-furylethylenes and 5-nitrofurans by using the comet assay in TK6 cells
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
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Abstract |
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The genotoxicity of three 2-furylethylene derivatives and four 5-nitrofurans was evaluated by using the comet assay in human lymphoblastoid cultured TK6 cells. The 2-furylethylene derivatives were 2-furyl-1-nitroethene, 1-(5-bromofur-2-yl)-2-nitroethene and 1-(5-bromofur-2-yl)-2-bromo-2-nitroethene, while the 5-nitrofurans were nitrofurantoin, nitrofurazone, furazolidone and 5-nitro-2-furanacrolein. The treatments lasted for 3 h in the absence of metabolic activation. No genotoxic effects were observed for two of the 2-furylethylene compounds, while the derivative 1-(5-bromofur-2-yl)-2-nitroethene showed a statistically significant response mainly at the highest concentration tested; this effect was considered biologically relevant and the compound was classified as slightly genotoxic. On the other hand, for the classical 5-nitrofurans tested there is a tendency towards a dose-related increase of the DNA damage in the comet assay and the observed increases for the parameters analysed (Olive tail moment, tail % DNA and tail length) were significant for all compounds. Then, the four 5-nitrofurans tested were considered genotoxic. These results show that the position of the nitro group influences the genotoxicity of the assayed compounds. Thus, in this comet assay, the 2-furylethylene derivatives having the nitro group attached outside the furan ring appear to be much less genotoxic than the 5-nitrofurans.
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Introduction |
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It has been shown that different 2-furylethylene derivatives possess interesting biological properties and potent microcidal activity (1
,2). Thus, these novel synthetic compounds have been proposed for their possible use in human and veterinary medicine.
Although these derivatives are not properly nitrofurans, the 2-furylethylene compounds with the nitro group located outside the furan ring are isomers of position of the classical 5-nitrofuran derivatives. Nevertheless, and contrary to the well-known genotoxicity of the 5-nitrofurans, the 2-furylethylene derivatives are considered not genotoxic (3). To test this hypothesis, our group has focused its efforts on the genotoxic evaluation of several 2-furylethylene derivatives by using different well-known in vitro and in vivo genotoxicity assays (4–8). The results of our studies showed the lack of genotoxicity in the in vitro and in vivo assays evaluating the induction of micronuclei. Nevertheless, slight increases in the sister chromatid exchanges (SCE) frequencies have been found mainly in the absence of the S9 microsomal fraction in the in vitro assays (4,5,7).
To confirm the different genotoxic potential of 2-furylethylene and 5-nitrofurans compounds, and bearing in mind their structural differences, mainly due to the position of the nitro group, we have measured the induction of DNA breakage by several compounds belonging to both families of chemicals.
DNA damage induction has been evaluated by means of the single cell gel electrophoresis assay, also called the comet test. This assay is considered a rapid, simple and sensitive technique for measuring DNA damage (9). In in vitro studies, it has emerged as a very sensitive method for detecting the genetic damage induced by different genotoxic agents such as radiation (10), pesticides (11) and arsenic compounds (12), as well as for examining DNA repair under a variety of experimental conditions (13). With the alkaline (pH > 13) version of the assay, effects such as DNA single-strand breaks (SSB), alkali-labile sites and SSB associated with incomplete excision repair sites can be easily analysed by the DNA that migrates out of the cell nucleus (9).
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Materials and methods |
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Cells used
The study was carried out using an established human lymphoblastoid cell line (TK6). This is a standard cell line with a stable karyotype, usually used in mutagenicity studies. The cells were maintained in suspension culture in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin and 2.5 IU/ml amphotericin B. The cultures were incubated at 37°C in a 5% CO2 incubator at 95% humidity.
Chemicals
The 2-furylethylene derivatives, 2-furyl-1-nitroethene (G-0, purity 96.38%), 1-(5-bromofur-2-yl)-2-nitroethene (2-ßNF, purity >96%) and 1-(5-bromofur-2-yl)-2-bromo-2-nitroethene (G-1, purity 99.53%) used in the study were synthesized at the Centro de Bioactivos Químicos (CBQ, Villa Clara, Cuba). The 5-nitrofurans used in the study were nitrofurantoin (NFT, purity >99%), nitrofurazone (NFA, purity >99%), furazolidone (FZ, purity 98%) and 5-nitro-2-furanacrolein (5-FAC, purity 97%) and were obtained from Sigma (Barcelona, Spain) (Figure 1).
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Treatments
Preliminary cytotoxicity assays were conducted using similar conditions to those used in the main study. They were carried out to determine the toxicity of the selected chemicals and to choose the concentrations to be used in the genotoxicity experiments.
Aliquots of 1 ml of cell suspension (106 TK6 cells) in RPMI 1640 medium were dispensed into eppendorf tubes. Two replicate cultures for concentration in each of the two experiments were established. All tested compounds were dissolved in dimethyl sulfoxide (DMSO, Panreac, Barcelona, Spain) just prior to treatment at the correct concentrations. DMSO was also tested as solvent control.
Aliquots of 10 µl of each treatment solution were added to the cultures and cells were incubated for 3 h at 37°C. The positive control group was treated with 10 µl of hydrogen peroxide (H2O2, Sigma, Barcelona, Spain) at a concentration of 2000 µM. After treatment, the cells were pelleted and washed twice in fresh RPMI 1640 medium and, finally, resuspended in 0.25 ml of fresh medium for the comet assay.
Cell viability was assessed, as soon as possible, after the treatments with a mix of fluorescein diacetate and ethidium bromide (EtBr) (14). Two hundred cells were scored for each concentration. The viability at the highest selected dose was >70%, which agrees with the usual testing conditions used and recommended for the comet assay (9,15).
Comet assay
The comet assay was performed, as previously described (16), with minor modifications. The cell samples (
40 000 cells in 20 µl) were carefully resuspended in 75 µl of 0.5% low melting point agarose (LMA), layered onto microscope slides pre-coated 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) and removal of the coverslip, 75 µl of 0.5% LMA was again added to the slides, covered and kept for 15–20 min at 4°C. Then the coverslips were removed and the slides were immersed 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 avoid 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 electrophoretic 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 were done 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 EtBr (0.4 µg/ml) just before the 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 BX50 fluorescence microscope equipped with a 480–550 nm wide band excitation filter and a 590 nm barrier filter. One hundred randomly selected cells (50 cells from each of the two replicate slides) were analysed per sample. The Olive tail moment, the tail percentage DNA and the tail length were used as measures of DNA damage and computed using the Komet version 3.1 software.
Statistical analysis
Only cells with a defined head were scored. The statistical analysis of the three parameters was done using the one-tailed Dunnett's test with the SPSS 11.5 program. Positive response was considered taking into account both biological and statistical relevance and a positive result was considered significant when P < 0.05. The mean, mean range and 95% confidence limits for the mean of the historical negative control from our laboratory were also considered in the interpretation of the results.
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Results |
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The results obtained in the induction of DNA damage following treatments with each of the selected chemicals are presented in Table I. TK6 cell treatments lasted for 3 h and were carried out without S9 metabolic fraction because, in previous studies, the 2-furylethylene compounds tested only increased the SCE frequency in these treatment conditions. To obtain comparable data, the same treatment conditions were used with both sets of compounds, 2-furylethylenes and 5-nitrofurans. Table I indicates the mean values for the Olive tail moment, tail % DNA and tail length from two different experiments for each evaluated concentration.
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The cell survival was assessed in preliminary experiments. For each compound and concentration tested, the viability was higher than 70% (data not showed). In general, the results for the three 2-furylethylene derivatives showed no excessive increase in DNA damage. The derivatives G-0 and G-1 were unable to induce DNA damage after the 3-h treatment at any of the assayed concentrations, when compared with the concurrent and historical vehicle controls. In both cases, positive controls showed clear and significant increases in the genetic damage measured.
For the compound 2-ßNF, statistically significant differences were observed after the treatment with the highest concentration tested for the tail % DNA and the Olive tail moment. For the tail length, significant increases were found for the three concentrations evaluated. The results obtained with the four classical 5-nitrofuran compounds showed, in general, a tendency towards a dose-related increase and significant increases in the induction of genetic damage taking into account the three parameters analysed.
NFT and 5-FAC induced DNA damage at the concentrations assayed, after treatments lasting for 3 h. Although in both cases, and at some concentrations, no statistically significant increases were found with respect to the mean values obtained in the controls, the results were considered biologically relevant when compared with the historical negative control data from our laboratory.
The results obtained with the two other 5-nitrofurans evaluated showed clear positive responses when compared with the concurrent and historical controls, for all the concentrations tested.
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Discussion |
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In previous experiments carried out in our laboratory with the derivatives G-0, G-1 and 2-ßNF, only slight increases in the frequency of SCE were observed in cultured human lymphocytes after treatments without metabolic activation (4
,5,7). Nevertheless, no induction of micronuclei was detected in such studies and, consequently, the tested compounds were considered as not genotoxic or very weakly genotoxic. To provide more data about the eventual genotoxic potential of these derivatives and to demonstrate a possible structure–activity relationship, we have performed the genotoxic evaluation of these compounds by using the comet assay and comparing the results with those obtained in the testing of classical 5-nitrofurans. Treatments were carried out without metabolic activation and, in the case of the 2-furylethylenes, whenever possible, the same range of concentrations applied in the previous SCE assays were used. The concentrations used for the 5-nitrofurans were established taking into account data reported in previous genotoxicity studies (17–20). Two of the three 2-furylethylene derivatives assayed (G-0 and G-1) have been unable to induce DNA damage as expressed by a significant increase in the parameters analysed and, as a consequence, they were considered non-genotoxic under the experimental conditions used in the comet assay. Although the G-1 compound showed a statistically significant increase in the tail % DNA at the two higher concentrations assayed, this was not considered biologically relevant since the values are between the 95% confidence limits of historical control data and are significantly fewer when compared with the mean observed for the historical negative control (Table II).
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In the case of the G-0 derivative, only a clear increase in the tail length was observed at the highest concentration tested, but it was lower than the top of the historical control range for the mean and the reached increase is only slightly higher when compared with the mean for the historical control and the top of the 95% confidence limits (Table II). Thus, the increase was considered to be without biological relevance.
The derivative (2-ßNF) showed a slight but statistically significant increase mainly at the highest concentration evaluated, and was considered slightly genotoxic in the present assay. The genotoxic effects were observed in all the parameters evaluated to determine a positive response in the DNA damage. Nevertheless, the increases were slightly higher when compared with the mean obtained in the historical control regarding the parameters analysed for the present comet assay (Table II).
With G-0 and 2-ßNF, we have used the same range of concentrations in the current comet study as in the previous SCE assays. Nevertheless, G-1 has proved to be more toxic in TK6 cells than in lymphocytes, hence the concentrations used in this work were lower.
The four 5-nitrofurans studied were considered genotoxic, including NFA and FZ, and they induced the highest increases in the DNA damage when compared with both concurrent and historical controls. These positive results are in concordance with the reported genotoxicity for the different 5-nitrofurans, including NFA and FZ. Thus, Anderson et al. (18) reported positive results in the incidence of chromosome aberrations for NFA and high increases in the percentage of aberrant cells in the absence of S9 mix at the concentration range 23–40 µg/ml. In addition, NFA caused a marked increase in the frequency of SCE at the concentration range 10–15 µg/ml that is close to the range used in the current study (5–30 µg/ml).
NFA and FZ were reported as strong mutagens on TA97 and TA102 strains of Salmonella typhimurium and induced the SOS-repair system in the PQ37 strain of Escherichia coli (21). Moreover, NFA showed clear evidence of carcinogenicity in female rats (mammary glands) and in female mice (ovary). NFA induced point mutations in Salmonella typhimurium and in mouse lymphoma L5178Y cells and also induced SCE and chromosome aberrations in Chinese hamster ovary cells (22).
FZ was reported as the most potent mutagen among eight nitrofurans tested in strains of S.typhimurium TA98, TA98NR and TA98/1,8-DNP6 (17). Significant increases in the frequency of SCE in cultures of human lymphocytes at the concentrations of 4–10 µg/ml were observed in an in-vitro study with FZ, as well as in in-vivo experiments (23).
The other two 5-nitrofurans studied, NFT and 5-FAC, were considered to be genotoxic in the present comet experiments at the concentrations assayed. Although, for both compounds, at some concentrations the increase observed for the tail % DNA and Olive tail moment was not statistically significant when compared with their concurrent control treatments, the values obtained for these parameters were significantly higher when compared with the mean for the historical control. On the other hand, the compounds showed significant increases in the tail length for all the concentrations evaluated. NFT has been reported to induce slight increases in the percentage of chromosome aberrations and SCE, in the absence of metabolic fractions, at concentrations of 25 and 50 µg/ml (18), as well as in the presence/absence of a metabolic fraction (22). In addition, NFT showed some evidence of carcinogenicity in male rats (kidney) and, in female mice, it showed clear evidence of carcinogenicity (ovary), as well as positive results in the induction of mutations in S.typhimurium and in the mouse lymphoma assay without metabolic activation.
Slapsyte et al. (19,20) published positive chromosome aberrations and SCE induction in in-vitro studies with human lymphocytes for NFT and furagin, a compound chemically and structurally similar to NFT. Both chromosome aberrations and SCE increases were observed in continuous 24-h treatments at concentrations of 20–40 µM, although high concentrations of furagin were required to induce chromosome aberrations in treatments lasting for 3 h.
The compound 5-FAC is a highly cytotoxic agent in the TK6 cultures showing the highest cytotoxicity among the 5-nitrofurans tested. 5-FAC was considered a potent mutagen in TA98, TA98NR and TA98/1,8-DNP6 S.typhimurium strains (17). Taking into account the present results in the comet assay, together with data from our previous studies, it seems that the compounds belonging to the series of 2-furylethylene derivatives, with the nitro group attached outside the furan ring, show lower genotoxic effects than the classical 5-nitrofurans compounds.
These results reinforce the view that the position of the nitro group gives to these two classes of nitro-compounds important differences in their genotoxicity. Thus, 2-furylethylene derivatives with the nitro group coupled outside the furan ring appear to be lower genotoxic agents when compared with the 5-nitrofurans. This would agree with several experimental data showing that only those derivatives with the nitro group in position 5 of the furan ring are mutagenic and the substitution of this functional group leads to the loss of mutagenic activity (3,24,25).
On the other hand, there is evidence for the possible detoxification or lower genotoxic effects of nitrofurans and 2-furylethylene derivatives in the presence of activating metabolic systems or in in vivo studies (4–7,20,21,24,26–28). For that reason, it may be possible to determine a safe dose range for this kind of 2-furylethylene derivatives for their therapeutic use. However, further in vivo genotoxicity studies are needed to elucidate the genetic safety of these chemicals.
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Acknowledgments |
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We would like to thank Dr Nilo Castañedo from CBQ (Villa Clara, Cuba) for kindly supplying the G-0, G-1 and 2-ßNF compounds. This investigation was supported in part by the Generalitat de Catalunya (2002SGR-00197).
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Notes |
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* To whom correspondence should be addressed. Tel: +34 93 581 2052; Fax: +34 93 581 2387; Email: ricard.marcos{at}uab.es
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References |
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Received on August 5, 2004; revised on September 27, 2004; accepted on March 15, 2005.
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