* Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; and
Centro de Investigación y Desarrollo Aplicado, Cida s.a.l., 08130 Santa Perpètua de Mogoda, Barcelona, Spain
Received November 7, 2002; accepted December 31, 2002
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ABSTRACT |
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Key Words: micronucleus assay; mice; furylethylenes; polychromatic erythrocytes; bone marrow.
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INTRODUCTION |
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Some nitro-compounds, among them the 5 nitrofurans, have showed genotoxicity and carcinogenicity in different experimental systems. Although there is also evidence that the furylethylene derivatives with the nitro group outside the furan ring do not show genotoxic effects (IARC, 1974; Jurado and Pueyo, 1995
; McCalla, 1983
; Ni et al., 1987
; Sturdik et al., 1985
). We have been studying a novel group of furylethylene derivatives, which have like common characteristic that the nitro group is attached in the exocyclic double bond of the ethylenic chain. In vitro studies, by using both the micronucleus (MN) and the sister chromatid-exchanges (SCE) test in cultured human lymphocytes, have been performed previously. These studies showed negative response in the micronucleus assay, with and without metabolic activation, and a slight increase in the SCE frequency has been observed, mainly in the absence of the S9 fraction. The slight effect disappears in the presence of the metabolic activation system (González Borroto et al., 2001
).
To obtain more insight into the genotoxic potential of this group of furylethylene derivatives, and to confirm the results obtained in the in vitro studies, an in vivo mutagenicity study has been carried out by using the mouse bone marrow-micronucleus test. In this study 2-furyl-1-nitroethene was selected as an example to assess the possible in vivo genotoxic potential of this chemical class, by measuring its effects on the formation of micronuclei in polychromatic erythrocytes (PCE) from the bone marrow of treated mice.
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MATERIAL AND METHODS |
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Animals were 67 weeks old when used for the determination of the doses in a preliminary dose-range-finding study; as well as in the main study for the micronucleus test. The test agent was dissolved in corn oil (Sigma-Aldrich, Spain) and the solution was administered intraperitoneally at the volume of 10 ml/kg of body weight. The negative control group received corn oil-only, while the positive control group was treated with cyclophosphamide (CP) at the concentration of 40 mg/kg, dissolved in physiologic solution.
In the preliminary dose-range finding experiment, animals were treated with G-0 in doses ranging from 10 to 100 mg/kg, and clinical signs of toxicity and deaths were recorded 24 h later. According to the symptoms of systemic toxicity elicited by G-0 and exhibited by animals, a series of three doses from 5 to 20 mg/kg was chosen to assess the micronuclei induction.
The micronucleus assay was conducted using a method based on the OECD and European Union guidelines (European Union, 1992; OECD, 1997
). Briefly, groups of five males were treated with G-0 (5, 10, or 20 mg/kg body weight). Only male mice were used, because in a previous study there was no marked sex-related difference in toxicity for this class of furylethylene derivatives (Ramos et al., 1997
). Separated groups of animals were killed at 24 and 48 h after dosing, by cervical dislocation. In the first sample time-period, animals dosed with the three doses (5, 10, or 20 mg/kg) were used; in the second sampling time, only animals treated with the high dose (20 mg/kg) were used. Femurs were removed and bone marrow collected in tubes containing 2 ml of fetal calf serum and centrifuged 5 min at 180 x g. Two smears were prepared and allowed to air dry, prior to fixation and staining with acridine orange solution. One drop of 0.04 mM acridine orange solution in Sörensen's phosphate buffer is placed on the fixed cells under a coverslip. Observations were made within a day by using an Olympus fluorescent microscope equipped with blue excitation and 515530 nm barrier filter (Krishna and Hayashi, 2000
).
Slides were coded and scored blind; 2000 polychromatic erythrocytes (PCEs) by animal were examined for the presence of micronuclei, which means 10,000 PCEs scored per dose-group. Due to the fact that normochromatic erythrocytes (NCE) have a lack of fluorescence or are viewed opaquely when the fluorescence stain was used, one slide more per animal was stained with May-Grünwald and Giemsa solutions. The ratio PCE/NCE was calculated by counting a total of 500 erythrocytes.
Statistical analysis.
The normality of the distribution of the frequency of micronucleated PCE (MNPCE/1000) scores was assessed by means of the Kolmogorov-Smirnov test of goodness of fit. The incidences of micronuclei in control and treated groups were then compared by using the Student's t-test for independent samples. The equality of variances was verified with Levene's test. The PCE/NCE ratio in control and treated groups was compared by the Student's t-test. Statistical decisions were made with a significance level of 0.05 (Crebelli et al., 1999; Jones et al., 2001
).
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RESULTS |
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In the main study, animals treated with the high dose (20 mg/kg), showed clinical signs of systemic toxicity, such as piloerection, palpebral ptosis, dyspnoea, and hunched posture. The symptoms confirmed the absorption of the test compound. Taking into account that bone marrow is a well-perfused tissue, and that levels of test substance in blood or plasma will be similar to those observed in bone marrow, we assumed that the test compound reached the target cells. Although, during the preliminary study, slides from animals dosed over the highest selected dose (20 mg/kg) were stained and observed, a reduction in the number of fluorescent PCEs was detected, indicating bone marrow toxicity. Besides, a decrease in the PCE/NCE ratio was obtained in a dose-dependent manner when the treatment and control groups were compared. The reduction is observed in both sampling times, and the values for doses of 10 and 20 mg/kg reached statistical significance.
The results of the micronucleus test with the furylethylene derivative selected for this study are summarized in Table 1. The incidence of MNPCE in each treatment group, the PCE/NCE ratio, as well as the number of micronucleated PCEs/scored PCEs are shown. Only one scorer analysed all the slides.
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The incidence of MNPCE in vehicle-treated mice (1.6 MNPCE/1000 PCEs) in both sampling times was within the accepted spontaneous range for this mouse strain (Krishna et al., 2000; Salamone and Mavournin, 1994
).
A statistically significant increase in the incidence of MNPCE over the control value was observed following treatment with the positive control substance cyclophosphamide (17.6 MNPCE/1000 PCE, P 0.001). This data confirmed the sensitivity of the experimental protocol followed in the detection of genotoxic effects.
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DISCUSSION |
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The G-0 compound is not properly a nitrofuran, but the furylethylene derivatives, with the nitro group located outside the furan ring, are isomers of position of the 5-nitrofurylethylene derivatives, and it has been indicated that such structural differences can produce changes in their genotoxic behaviour (Estrada, 1998). It has also been reported that only those derivatives with the nitro group coupled in position 5 of the furan ring are mutagenic, and that the substitution of this functional group results in the loss of mutagenic activity (Estrada, 1998
; González Borroto et al., 2001
; McCalla, 1983
; Sturdik et al., 1985
). In addition, several studies confirm the possible detoxification of nitrofurans when they are evaluated in the presence of activating metabolic systems or in in vivo studies (Gajewska et al., 1990
; Hatcher et al., 1995
; Murthy and Najaria, 1980
; Skeggs et al., 1984
).
In a previous study carried out in our laboratory, the in vitro genotoxicity of the G-0 was evaluated in cultured human lymphocytes. This derivative did not demonstrate any capacity to induce micronuclei, with and without S9 mix, by using the cytokinesis-block micronucleus technique, although it was considered slightly genotoxic in the sister-chromatid exchanges (SCE) assay without S9 (González Borroto et al., 2001). We have obtained similar results when the in vitro genotoxic effects of other two furylethylene derivatives, with very similar chemical structure [1-(5-bromofur-2-yl)-2-bromo-2-nitroethene (G-1) and 1-(5-bromofur-2-yl)-2-nitroethene (2-bNF)], have been evaluated (González Borroto et al., 2002
). The results of theses studies are summarized in Table 2
.
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In the present study, no effects on the induction of MNPCE were observed with the test agent in a range of doses from 5 to 20 mg/kg in treated mice. Our previous in vitro results and the in vivo results reported here, as well as the other data mentioned above, would indicate/confirm the detoxification exerted by the metabolism on the possible genotoxicity of this class of chemicals. On the other hand, the global results of in vitro and in vivo studies with the test agent G-0, and other related derivatives, indicate the possibility that there is a safety margin for the use of these novel compounds in human and veterinary medicine. Nevertheless, additional mutagenicity studies measuring different levels of DNA damage are still necessary.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Estrada, E. (1998). Structure-mutagenicity relationships in 2-furylethylene derivatives. A molecular orbital study of the role of nitro groups. Mutat. Res. 420, 6775.[ISI][Medline]
European Union (1992). Commission Directive 92/69/EEC, Annex V B12. Off. J. Eur. Community 35, 154156.
Gajewska, J., Szczypka, M., Tudek, B., and Szymczyk, T. (1990). Studies on the effect of ascorbic acid and selenium on the genotoxicity of nitrofurans: Nitrofurazone and furazolidone. Mutat. Res. 232, 191197.[CrossRef][ISI][Medline]
González Borroto, J. G., Creus, A., and Marcos, R. (2001). Genotoxic evaluation of the furylethylene derivative 2-furyl-1-nitroethene in cultured human lymphocytes. Mutat. Res. 497, 177184.[ISI][Medline]
González Borroto, J. G., Creus, A., and Marcos, R. (2002). Genotoxic evaluation of the furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene in cultured human lymphocytes. Mutat. Res. 519, 179185.[ISI][Medline]
Hamilton-Miller, J. M. T. (1997). Nitrofurans. In Antibiotic and Chemotherapy, 7th ed. (F. O'Grady, H. P. Lambert, R. G. Finch, and D. Greenwood, Eds.), pp. 396403. Churchill Livingstone, London.
Hatcher, J. F., Yamamoto, K., Ichikawa, M., Bryan, G. T., and Swaminathan, S. (1995). Metabolic reduction of novel 3,4-dichloro-5-nitrofurans in Salmonella typhimurium. Environ. Mol. Mutagen. 25, 5866.[ISI][Medline]
IARC (1974). International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 7, Some Anti-Thyroid and Related Substances, Nitrofurans and Industrial Chemicals. IARC, Lyon, France.
Jones, E., Fox, V., Elliott, B. M., and Moore, N. P. (2001). The mutagenic potential of acetonitrile in the bone marrow and peripheral blood of the mouse. Mutagenesis 2, 151154.[CrossRef]
Jurado, J., and Pueyo, C. (1995). Role of classical nitroreductase and O-acetyltransferase on the mutagenicity of nifurtimox and eight derivatives in Salmonella typhimurium. Environ. Mol. Mutagen. 26, 8693.[ISI][Medline]
Krishna, G., and Hayashi, M. (2000). In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat. Res. 455, 155166.[ISI][Medline]
Krishna, G., Urda, G., and Paulissen, J. (2000). Historical vehicle and positive control micronucleus data in mice and rats. Mutat. Res. 453, 4550.[ISI][Medline]
McCalla, D. R. (1983). Mutagenicity of nitrofuran derivatives: Review. Environ. Mutagen. 5, 745765.[ISI][Medline]
Murthy, M. S. S., and Najaria, K. B. (1980). Deactivation of furyl furamide (AF-2) by rat-liver microsomes and its implication in short-term test for mutagenicity/carcinogenicity. Mutat. Res. 77, 127134.[ISI][Medline]
Ni, Y. C., Heflich, R. H., Kadlubar, F. F., and Fu, P. P. (1987). Mutagenicity of nitrofurans in Salmonella typhimurium TA98, TA98NR, and TA98/1,8-DNP6. Mutat. Res. 192, 1522.[CrossRef][ISI][Medline]
OECD (1997). Guideline 474, Mammalian Erythrocyte Micronucleus Test, OECD Guideline for the Testing of Chemicals, pp. 110. OECD, Paris, France.
Ramos, A., Vizoso, A., Edreira, A., Betancourt, J., and Decalo, M. (1997). Activity of nitroalkenederivative 1-(5-bromofur-2-il)-2-bromo-2-nitroethene in Salmonella/microsome assay and the mouse bone marrow micronucleus test. Mutat. Res. 390, 233238.[ISI][Medline]
Salamone, M. F., and Mavournin, K. H. (1994). Bone marrow micronucleus assay: A review of the mouse stocks used and their published mean spontaneous micronucleus frequencies. Environ. Mol. Mutagen. 23, 239273.[ISI][Medline]
Skeggs, H. R., Berglund, R. M., VandenHeuvel, W. J. A., Mrozik, H., Wislocki, P. G., and Wolf, F. J. (1984). Effect of liver enzymes on the mutagenicity of nitroheterocyclic compounds: Activation of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1-H-imidazol-2-yl)-1,2-benzisoxazole and deactivation of nitrofurans and nitroimidazoles in the Ames test. Mutat. Res. 136, 18.[ISI][Medline]
Slapsyte, G., Jankauskiene, A., Mierauskiene, J., and Lazutka, J. R. (2002). Cytogenetic analysis of peripheral blood lymphocytes of children treated with nitrofurantoin for recurrent urinary tract infection. Mutagenesis. 1, 3135.[CrossRef]
Sturdik, E., Rosenberg, M., Stibranyi, L., Balaz, S., Chreno, O., Ebringer, L., Ilavsky, D., and Vegh, D. (1985). Structure-mutagenicity relationships of 5-nitro-2-furylethylenes in Salmonella typhimurium TA 98. Chem. Biol. Interact. 53, 145153.[ISI][Medline]