This article was downloaded by: [Universite Laval] On: 10 October 2014, At: 12:22 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Genotoxic and Mutagenic Effects of Diflubenzuron, an Insect Growth Regulator, on Mice a
a
b
Aline Lima de Barros , Vanessa Vilamaior de Souza , Stephanie Dynczuki Navarro , Silvia a
b
a
Aparecida Oesterreich , Rodrigo Juliano Oliveira , Cândida Aparecida Leite Kassuya & Arielle Cristina Arena
a c
a
School of Health Sciences, Federal University of Grande Dourados (UFGD) , Dourados , Mato Grosso do Sul State , Brazil b
Federal University of Mato Grosso do Sul (UFMS)–Campo Grande, Campo Grande , Mato Grosso do Sul State , Brazil c
Department of Morphology , Institute of Biosciences of Botucatu, São Paulo State University (UNESP) , Botucatu , São Paulo State , Brazil Published online: 29 Oct 2013.
To cite this article: Aline Lima de Barros , Vanessa Vilamaior de Souza , Stephanie Dynczuki Navarro , Silvia Aparecida Oesterreich , Rodrigo Juliano Oliveira , Cândida Aparecida Leite Kassuya & Arielle Cristina Arena (2013) Genotoxic and Mutagenic Effects of Diflubenzuron, an Insect Growth Regulator, on Mice, Journal of Toxicology and Environmental Health, Part A: Current Issues, 76:17, 1003-1006, DOI: 10.1080/15287394.2013.830585 To link to this article: http://dx.doi.org/10.1080/15287394.2013.830585
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 76:1003–1006, 2013 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2013.830585
GENOTOXIC AND MUTAGENIC EFFECTS OF DIFLUBENZURON, AN INSECT GROWTH REGULATOR, ON MICE Aline Lima de Barros1, Vanessa Vilamaior de Souza1, Stephanie Dynczuki Navarro2, Silvia Aparecida Oesterreich1, Rodrigo Juliano Oliveira2, Cândida Aparecida Leite Kassuya1, Arielle Cristina Arena1,3 1
Downloaded by [Universite Laval] at 12:22 10 October 2014
School of Health Sciences, Federal University of Grande Dourados (UFGD), Dourados, Mato Grosso do Sul State, Brazil 2 Federal University of Mato Grosso do Sul (UFMS)–Campo Grande, Campo Grande, Mato Grosso do Sul State, Brazil 3 Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Botucatu, São Paulo State, Brazil This study assessed the genotoxic and mutagenic potential of diflubenzuron (DFB) insecticide in mice. Mice were divided into five groups: group I: negative control; group II: positive control; group III: 0.3 mg/kg of DFB; group IV: 1 mg/kg of DFB; group V: 3 mg/kg DFB. Peripheral blood was collected for the comet assay and the micronucleus (MN) test. DFB increased incidence of comet formation at all doses tested. A rise in the frequency of MN in mouse peripheral blood was observed 24, 48, and 72 h postexposure at all doses tested. Data demonstrate that DFB exerts genotoxic and mutagenic effects in a dose-dependent manner.
not clear whether DFB as a consequence of its presence in the environment may induce genotoxicity in nontarget species. Determination of the genotoxic potential of environmental pollutants aims to assess risks to genetic material, not only in exposed humans but also to specific native biota at the spraying sites (Pierce et al., 1998). The “Guidelines for Carcinogen Risk Assessment” published by the U.S. Environmental Protection Agency point out that changes in gene expression, DNA repair, cell cycle control, genomic instability, and chromosomal damage are the main consequences of exposure to environmental agents and critical events involved in carcinogenesis (Preston, 2007). Given that DFB is an important insecticide widely used to control the Aedes aegypti larvae, this study aims to assess the genotoxic and mutagenic potential of this
Diflubenzuron (DFB), 1-(4-chlorophenyl)3-(2,6-difluorobenzoyl) urea, is an insecticide and acaricide insect growth regulator (IRC) belonging to the class of benzoylureas, and is used in (1) agriculture against insect predators, (2) fisheries to control fish ectoparasites, and (3) public health programs to control insects and vectors, mainly Aedes aegypti larvae (Agencia Nacional de Vigilância Sanitária [ANVISA], 1985; United Nations Environment Programme, International Labour Organisation, 1996). This class of insecticides acts by interfering with the deposition of chitin, one of the components of the insect cuticle (Reynolds, 1987), and is most effective on immature forms (larvae). The mechanism of action of these compounds is to prevent the insect from releasing itself from the exocuticle, particularly during ecdysis (Silva et al., 2003). At present, it is
Received 29 July 2013; accepted 29 July 2013. The authors thank CAPES for the financial assistance and Champion Farmoquimica LTDA for providing of insecticide. Address correspondence to Arielle Cristina Arena, Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Distrito de Rubião Junior, s/n, Caixa Postal–510, CEP 18618970 Botucatu, SP, Brazil. E-mail: ariellearena@ibb. unesp.br 1003
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A. L. DE BARROS ET AL.
compound in mice exposed to different doses and to examine alterations in nontarget organisms under controlled lab conditions.
MATERIALS AND METHODS
Downloaded by [Universite Laval] at 12:22 10 October 2014
Animals and Treatment Adult male Swiss mice (60 d old, weighing 30–40 g, n = 50) from the Federal University of Grande Dourados were maintained under controlled conditions (22 ± 2◦ C, 50% humidity, 12-h light/dark cycle). Mice were identified individually, and each group was housed in polypropylene cages with filtered water and commercial food ad libitum. This study was approved by the Ethical Committee of the UNESP (São Paulo State University, SP, Brazil) (protocol 380/2012). Animals were divided into five groups (n = 10/group). Three groups received diflubenzuron (DFB; 1-(4-chlorophenyl)3-(2.6-difluorobenzoyl) urea; Champion Farmoquímica Ltda, purity level of 98%), at doses of 0.3 (group III), 1 (group IV), or 3 (group V) mg/kg body weight by oral route (gavage). The dose regimens were based on a no-observed-adverse-effect level (NOAEL) of DFB (2 mg/kg body weight/d; EHC 184, 1996). The negative control animals (group I) were exposed only to corn oil (vehicle). The positive control animals (group II) were treated with cyclophosphamide (Fosfaseron, Filaxis) at a dose of 100 mg/kg. The treatment was performed once, and DFB was dissolved in corn oil and cyclophosphamide was dissolved in saline before administration. Comet Assay and Micronucleus (MN) Test With Peripheral Blood Comet assay Twenty-four hours after treatment with DFB, 40 µl blood was collected from each animal in each group, mixed with 120 µl of agarose LPM (1.5%) at 37◦ C, and placed on the slide. Slides were precoated with 5% agarose. Coverslips were removed and slides were immersed in lysis solution freshly prepared with 89 ml lysis stock solution (2.5
M NaCl, 100 mM ethylenediamine tetraacetic acid [EDTA], 10 mM Tris, pH 10 adjusted with NaOH, 89 ml distilled water, and 1% of sodium lauroylsarcosine), 1 ml Triton X-100, and 10 ml dimethyl sulfoxide (DMSO), for 1 h at 4◦ C in the dark. Slides were placed in an electrophoresis chamber filled with buffer, pH > 13 (300 mM NaOH and 1 mM EDTA, prepared with a stock solution of 10 N NaOH and EDTA 200 mM, pH 10), at 4◦ C for 20 min for DNA denaturation. Electrophoresis was performed at 25 V and 300 mA (1.25 V/cm). Slides were then neutralized with buffer pH 7.5 (0.4 M Tris-HCl) during 3 cycles of 5 min, airdried, and fixed with 100% ethanol for 10 min. Slides were stained with 100 µl of ethidium bromide (EB; 20 µg/ml) and slip covered. The material was examined with a fluorescence microscope, equipped with an excitation filter of 515–560 nm and a barrier filter of 520 nm at 40× magnification. One hundred cells per animal were examined and comets were classified according to De Oliveira et al. (2010). A total damage score was determined by multiplying the number of cells assigned to each class of damage by the numeric value of the class and summing all resulting values. Micronucleus test The micronucleus (MN) test with peripheral blood was conducted according to Hayashi et al. (1990). Blood was collected from the tail of animals from all groups at 24, 48, and 72 h after treatment. Slides were previously prepared with 20 µl acridine orange, and 20 µl peripheral blood was added in the center of prepared slides. Slides were slip covered and stored in a freezer. Examination of slides was conducted with a fluorescence microscope under blue light (488 nm), an orange barrier filter, and magnification of 100×. Two thousand cells per animal (two slides per animal per treatment) were examined and micronucleated cells were counted. The frequency of MN was examined at 24 , 48, and 72 h after treatment. Statistical Analysis Data were subjected to analysis of variance (ANOVA). Significant differences were
GENOTOXIC AND MUTAGENIC EFFECTS OF DIFLUBENZURON
determined using Tukey’s test. The criterion for significance was set at p < .05.
1005
approximately 22.73-, 27.9- and 21-fold at 24, 48, and 72 hr, respectively. DISCUSSION
Downloaded by [Universite Laval] at 12:22 10 October 2014
RESULTS
One of the metabolites of DFB, 4chloroaniline, was classified by the U.S. Environmental Protection Agency (EPA) as a probable human carcinogen (U.S. EPA, 1997), which reinforces the need for genotoxic evaluation of this compound. This study demonstrated that the pesticide DFB induced DNA damage in mice at the doses tested. In the present study, the genotoxic effects of DFB were evaluated using the comet assay, which detects DNA damage produced by chemical and physical agents (Gontijo and Tice, 2003). Thus, DFB increased the incidence of comets at all doses tested, with a greater number of damaged cells at doses of 1 and 3 mg/kg, indicating dose-dependent genotoxicity. The incidence of damaged cells found in the groups treated with DFB was higher than those found in the positive control group, which received cyclophosphamide. In this study, cyclophosphamide was used to induce DNA damage, which has already proven genotoxic activity in bone marrow cells and peripheral blood in vivo and in vitro (Fenech et al. 1999). The mutagenic effects of DFB were examined using the MN test in peripheral blood, where it is possible to detect changes in DNA and/or damage to the spindle (Fenech, 2000). After treatment with DFB there was an increase in the incidence of MN in peripheral blood of mice at all doses tested, whereas the highest
Table 1 presents the overall frequency of damaged cells, the division between the classes of damage, and score ± SEM for the comet assay. The frequency of genotoxic damage indicates that DFB has genotoxic potential similar to cyclophosphamide, which was used as positive control. Cyclophosphamide elevated the frequency of comets by 3.5-fold. The doses of 0.3, 1, and 3 mg/kg of DFB increased the frequency in the order of 3.6-, 3.69-, and 3.77fold, respectively. Cyclophosphamide and the lowest dose of DFB exhibited similar scores and showed a rise of approximately 4.37- and 4.16-fold, respectively. Regarding the extent of genetic damage, it was found that the two highest doses of DFB elevated damage by 5.47and 5.07-fold at 1 and 3 mg/kg of DFB, respectively. With regards to the frequency of MN (Table 2), all DFB doses tested were mutagenic. Cyclophosphamide increased the frequency of MN by 14.33-, 15-, and 10.78- fold at 24, 48, and 72 h, respectively. When the different DFB doses were evaluated it was observed that the two lower doses showed mutagenic activity similar to that of cyclophosphamide, and that the highest dose was further increased. Results indicate, therefore, that the mutagenic/toxic ability 3 mg/kg was higher than that of cyclophosphamide and MN frequency rose to TABLE 1. Frequency of Damage, by the Comet Assay Damage classes Treatments
Damaged cells
0
1
2
3
Score
Negative control Positive control DFB 0.3mg/kg (GI) DFB 1.0 mg/kg (GII) DFB 3.0 mg/kg (GIII)
24.20 ± 2.88a 88.40 ± 1.71b 89.40 ± 1.01b 91.50 ± 0.52b 93.50 ± 0.65b
75.80 ± 2.88 11.30 ± 1.80 10.60 ± 1.01 8.40 ± 0.50 6.50 ± 0.65
20.80 ± 2.52 61.10 ± 2.81 66.20 ± 2.27 49.30 ± 1.22 43.20 ± 1.21
3.20 ± 0.55 21.50 ± 2.19 20.00 ± 1.99 34.80 ± 1.64 41.90 ± 1.16
0.20 ± 1.33 5.80 ± 0.78 3.20 ± 0.70 7.40 ± 0.54 8.40 ± 0.83
27.80 ± 3.34a 121.50 ± 4.34b 115.80 ± 2.67b 141.10 ± 2.02c 152.20 ± 2.17c
Note. Data presented as mean ± SEM. Different letters indicate statistically significant differences (p < .05; ANOVA/Tukey). Class 0, no damage; class 1, tail of comet shorter than the diameter of nucleiod; class 2, tail of comet once or twice the diameter of nucleoid; class 3, tail of comet more than twice the diameter of nucleoid.
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A. L. DE BARROS ET AL.
TABLE 2. Frequency of Micronuclei Time (h) Treatments
24
48
72
Negative control Positive control DFB 0.3 mg/kg (GI) DFB 1 mg/kg (GII) DFB 3 mg/kg (GIII)
1.50 ± 0.65a 21.50 ± 1.94b 16.80 ± 1.58b 17.60 ± 1.66b 34.10 ± 2.16c
1.00 ± 0.37a 15.00 ± 1.19b 11.40 ± 1.16b 16.30 ± 1.42b 27.90 ± 1.62c
0.90 ± 0.31a 9.70 ± 0.40b 9.80 ± 0.79b 12.40 ± 0.96b 18.90 ± 1.33c
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Note. Data presented are mean ± SEM, frequency of micronuclei per 2000 cells. Different letters indicate statistically significant differences, ANOVA/Tukey (p < .05).
dose (3 mg/kg) showed a significantly higher incidence of MN than the positive control group (cyclophosphamide). Data demonstrate that DFB exerts mutagenic effects in a dosedependent manner. It was found that the frequency of MN decreased chronologically after treatment. This decrease may be due to metabolism of DFB which then reduces its ability to induce DNA damage. Data demonstrated that DFB possesses genotoxic and mutagenic capacity to affect nontarget organisms in a dose-related manner. REFERENCES Agencia Nacional de Vigilância Sanitária. 1985. Índice Monográfico. D17 Diflubenzuron. http://portal.anvisa.gov.br/wps/wcm/ connect/af1db28047458f 8d98c8dc3fbc4c 6735/d17_novo.pdf?MOD=AJPERES&use DefaultText=0&useDefaultDesc=0. De Oliveira, P. R., Bechara, G. H., Denardi, S. E., Oliveira, R. J., and Mathias, M. I. C. 2010. Genotoxic and mutagenic effects of fipronil on mice. Exp. Toxicol. Pathol. 64: 569–777. Fenech, M. 2000. The in vitro micronucleus technique. Mutat. Res. 455: 81–95. Fenech, M., Holland, N., Chang, W. P., Zeiger, E., and Bonassi, S. 1999. The human micronucleus project—An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutat. Res. 428: 271–283. Gontijo, A. M. M. C., and Tice, R. 2003. Teste do cometa para a detecção de dano no
DNA e reparo em células individualizadas. In Mutagênese ambiental, ed. L. R. Ribeiro, E. K. Salvadori, and E. K. Marques, 247–275. Canoas, Brazil: ULBRA. Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, M., Jr. 1990. The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. Mutat. Res. 245: 245–249. Peirce, J. J., Weiner, R. F., and Vesilind, P. A. 1998. Meteorology and air pollution. In Environmental pollution and control, 271–286. Boston, MA: ButterworthHeinemann. Preston, R. J. 2007. Epigenetic processes and cancer risk assessment. Mutat. Res. 616: 7–10. Reynolds, S. E. 1987. The cuticule, growth regulators and moulting in insects: The essential background to the action of acylurea insecticides. Pestic. Sci. 20: 131–146. Silva, M. T. B., Costa, E. C., and Boss, A. 2003. Control of Anticarsiagemmatalis Hübner (Lepidoptera: Noctuidae) larvae with insect growth regulators. Cienc. Rural. 33: 601–605. United Nations Environment Programme, International Labour Organisation. 1996. Environmental health criteria 184. Diflubenzuron. Geneva, Switzerland: World Health Organization. U.S. Environmental Protection Agency. 1997. Reregistration eligibility decision— Diflubenzuron. Washington, DC: Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA.
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Genotoxic and Mutagenic Effects of Diflubenzuron, an Insect Growth Regulator, on Mice a
a
b
Aline Lima de Barros , Vanessa Vilamaior de Souza , Stephanie Dynczuki Navarro , Silvia a
b
a
Aparecida Oesterreich , Rodrigo Juliano Oliveira , Cândida Aparecida Leite Kassuya & Arielle Cristina Arena
a c
a
School of Health Sciences, Federal University of Grande Dourados (UFGD) , Dourados , Mato Grosso do Sul State , Brazil b
Federal University of Mato Grosso do Sul (UFMS)–Campo Grande, Campo Grande , Mato Grosso do Sul State , Brazil c
Department of Morphology , Institute of Biosciences of Botucatu, São Paulo State University (UNESP) , Botucatu , São Paulo State , Brazil Published online: 29 Oct 2013.
To cite this article: Aline Lima de Barros , Vanessa Vilamaior de Souza , Stephanie Dynczuki Navarro , Silvia Aparecida Oesterreich , Rodrigo Juliano Oliveira , Cândida Aparecida Leite Kassuya & Arielle Cristina Arena (2013) Genotoxic and Mutagenic Effects of Diflubenzuron, an Insect Growth Regulator, on Mice, Journal of Toxicology and Environmental Health, Part A: Current Issues, 76:17, 1003-1006, DOI: 10.1080/15287394.2013.830585 To link to this article: http://dx.doi.org/10.1080/15287394.2013.830585
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 76:1003–1006, 2013 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2013.830585
GENOTOXIC AND MUTAGENIC EFFECTS OF DIFLUBENZURON, AN INSECT GROWTH REGULATOR, ON MICE Aline Lima de Barros1, Vanessa Vilamaior de Souza1, Stephanie Dynczuki Navarro2, Silvia Aparecida Oesterreich1, Rodrigo Juliano Oliveira2, Cândida Aparecida Leite Kassuya1, Arielle Cristina Arena1,3 1
Downloaded by [Universite Laval] at 12:22 10 October 2014
School of Health Sciences, Federal University of Grande Dourados (UFGD), Dourados, Mato Grosso do Sul State, Brazil 2 Federal University of Mato Grosso do Sul (UFMS)–Campo Grande, Campo Grande, Mato Grosso do Sul State, Brazil 3 Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Botucatu, São Paulo State, Brazil This study assessed the genotoxic and mutagenic potential of diflubenzuron (DFB) insecticide in mice. Mice were divided into five groups: group I: negative control; group II: positive control; group III: 0.3 mg/kg of DFB; group IV: 1 mg/kg of DFB; group V: 3 mg/kg DFB. Peripheral blood was collected for the comet assay and the micronucleus (MN) test. DFB increased incidence of comet formation at all doses tested. A rise in the frequency of MN in mouse peripheral blood was observed 24, 48, and 72 h postexposure at all doses tested. Data demonstrate that DFB exerts genotoxic and mutagenic effects in a dose-dependent manner.
not clear whether DFB as a consequence of its presence in the environment may induce genotoxicity in nontarget species. Determination of the genotoxic potential of environmental pollutants aims to assess risks to genetic material, not only in exposed humans but also to specific native biota at the spraying sites (Pierce et al., 1998). The “Guidelines for Carcinogen Risk Assessment” published by the U.S. Environmental Protection Agency point out that changes in gene expression, DNA repair, cell cycle control, genomic instability, and chromosomal damage are the main consequences of exposure to environmental agents and critical events involved in carcinogenesis (Preston, 2007). Given that DFB is an important insecticide widely used to control the Aedes aegypti larvae, this study aims to assess the genotoxic and mutagenic potential of this
Diflubenzuron (DFB), 1-(4-chlorophenyl)3-(2,6-difluorobenzoyl) urea, is an insecticide and acaricide insect growth regulator (IRC) belonging to the class of benzoylureas, and is used in (1) agriculture against insect predators, (2) fisheries to control fish ectoparasites, and (3) public health programs to control insects and vectors, mainly Aedes aegypti larvae (Agencia Nacional de Vigilância Sanitária [ANVISA], 1985; United Nations Environment Programme, International Labour Organisation, 1996). This class of insecticides acts by interfering with the deposition of chitin, one of the components of the insect cuticle (Reynolds, 1987), and is most effective on immature forms (larvae). The mechanism of action of these compounds is to prevent the insect from releasing itself from the exocuticle, particularly during ecdysis (Silva et al., 2003). At present, it is
Received 29 July 2013; accepted 29 July 2013. The authors thank CAPES for the financial assistance and Champion Farmoquimica LTDA for providing of insecticide. Address correspondence to Arielle Cristina Arena, Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Distrito de Rubião Junior, s/n, Caixa Postal–510, CEP 18618970 Botucatu, SP, Brazil. E-mail: ariellearena@ibb. unesp.br 1003
1004
A. L. DE BARROS ET AL.
compound in mice exposed to different doses and to examine alterations in nontarget organisms under controlled lab conditions.
MATERIALS AND METHODS
Downloaded by [Universite Laval] at 12:22 10 October 2014
Animals and Treatment Adult male Swiss mice (60 d old, weighing 30–40 g, n = 50) from the Federal University of Grande Dourados were maintained under controlled conditions (22 ± 2◦ C, 50% humidity, 12-h light/dark cycle). Mice were identified individually, and each group was housed in polypropylene cages with filtered water and commercial food ad libitum. This study was approved by the Ethical Committee of the UNESP (São Paulo State University, SP, Brazil) (protocol 380/2012). Animals were divided into five groups (n = 10/group). Three groups received diflubenzuron (DFB; 1-(4-chlorophenyl)3-(2.6-difluorobenzoyl) urea; Champion Farmoquímica Ltda, purity level of 98%), at doses of 0.3 (group III), 1 (group IV), or 3 (group V) mg/kg body weight by oral route (gavage). The dose regimens were based on a no-observed-adverse-effect level (NOAEL) of DFB (2 mg/kg body weight/d; EHC 184, 1996). The negative control animals (group I) were exposed only to corn oil (vehicle). The positive control animals (group II) were treated with cyclophosphamide (Fosfaseron, Filaxis) at a dose of 100 mg/kg. The treatment was performed once, and DFB was dissolved in corn oil and cyclophosphamide was dissolved in saline before administration. Comet Assay and Micronucleus (MN) Test With Peripheral Blood Comet assay Twenty-four hours after treatment with DFB, 40 µl blood was collected from each animal in each group, mixed with 120 µl of agarose LPM (1.5%) at 37◦ C, and placed on the slide. Slides were precoated with 5% agarose. Coverslips were removed and slides were immersed in lysis solution freshly prepared with 89 ml lysis stock solution (2.5
M NaCl, 100 mM ethylenediamine tetraacetic acid [EDTA], 10 mM Tris, pH 10 adjusted with NaOH, 89 ml distilled water, and 1% of sodium lauroylsarcosine), 1 ml Triton X-100, and 10 ml dimethyl sulfoxide (DMSO), for 1 h at 4◦ C in the dark. Slides were placed in an electrophoresis chamber filled with buffer, pH > 13 (300 mM NaOH and 1 mM EDTA, prepared with a stock solution of 10 N NaOH and EDTA 200 mM, pH 10), at 4◦ C for 20 min for DNA denaturation. Electrophoresis was performed at 25 V and 300 mA (1.25 V/cm). Slides were then neutralized with buffer pH 7.5 (0.4 M Tris-HCl) during 3 cycles of 5 min, airdried, and fixed with 100% ethanol for 10 min. Slides were stained with 100 µl of ethidium bromide (EB; 20 µg/ml) and slip covered. The material was examined with a fluorescence microscope, equipped with an excitation filter of 515–560 nm and a barrier filter of 520 nm at 40× magnification. One hundred cells per animal were examined and comets were classified according to De Oliveira et al. (2010). A total damage score was determined by multiplying the number of cells assigned to each class of damage by the numeric value of the class and summing all resulting values. Micronucleus test The micronucleus (MN) test with peripheral blood was conducted according to Hayashi et al. (1990). Blood was collected from the tail of animals from all groups at 24, 48, and 72 h after treatment. Slides were previously prepared with 20 µl acridine orange, and 20 µl peripheral blood was added in the center of prepared slides. Slides were slip covered and stored in a freezer. Examination of slides was conducted with a fluorescence microscope under blue light (488 nm), an orange barrier filter, and magnification of 100×. Two thousand cells per animal (two slides per animal per treatment) were examined and micronucleated cells were counted. The frequency of MN was examined at 24 , 48, and 72 h after treatment. Statistical Analysis Data were subjected to analysis of variance (ANOVA). Significant differences were
GENOTOXIC AND MUTAGENIC EFFECTS OF DIFLUBENZURON
determined using Tukey’s test. The criterion for significance was set at p < .05.
1005
approximately 22.73-, 27.9- and 21-fold at 24, 48, and 72 hr, respectively. DISCUSSION
Downloaded by [Universite Laval] at 12:22 10 October 2014
RESULTS
One of the metabolites of DFB, 4chloroaniline, was classified by the U.S. Environmental Protection Agency (EPA) as a probable human carcinogen (U.S. EPA, 1997), which reinforces the need for genotoxic evaluation of this compound. This study demonstrated that the pesticide DFB induced DNA damage in mice at the doses tested. In the present study, the genotoxic effects of DFB were evaluated using the comet assay, which detects DNA damage produced by chemical and physical agents (Gontijo and Tice, 2003). Thus, DFB increased the incidence of comets at all doses tested, with a greater number of damaged cells at doses of 1 and 3 mg/kg, indicating dose-dependent genotoxicity. The incidence of damaged cells found in the groups treated with DFB was higher than those found in the positive control group, which received cyclophosphamide. In this study, cyclophosphamide was used to induce DNA damage, which has already proven genotoxic activity in bone marrow cells and peripheral blood in vivo and in vitro (Fenech et al. 1999). The mutagenic effects of DFB were examined using the MN test in peripheral blood, where it is possible to detect changes in DNA and/or damage to the spindle (Fenech, 2000). After treatment with DFB there was an increase in the incidence of MN in peripheral blood of mice at all doses tested, whereas the highest
Table 1 presents the overall frequency of damaged cells, the division between the classes of damage, and score ± SEM for the comet assay. The frequency of genotoxic damage indicates that DFB has genotoxic potential similar to cyclophosphamide, which was used as positive control. Cyclophosphamide elevated the frequency of comets by 3.5-fold. The doses of 0.3, 1, and 3 mg/kg of DFB increased the frequency in the order of 3.6-, 3.69-, and 3.77fold, respectively. Cyclophosphamide and the lowest dose of DFB exhibited similar scores and showed a rise of approximately 4.37- and 4.16-fold, respectively. Regarding the extent of genetic damage, it was found that the two highest doses of DFB elevated damage by 5.47and 5.07-fold at 1 and 3 mg/kg of DFB, respectively. With regards to the frequency of MN (Table 2), all DFB doses tested were mutagenic. Cyclophosphamide increased the frequency of MN by 14.33-, 15-, and 10.78- fold at 24, 48, and 72 h, respectively. When the different DFB doses were evaluated it was observed that the two lower doses showed mutagenic activity similar to that of cyclophosphamide, and that the highest dose was further increased. Results indicate, therefore, that the mutagenic/toxic ability 3 mg/kg was higher than that of cyclophosphamide and MN frequency rose to TABLE 1. Frequency of Damage, by the Comet Assay Damage classes Treatments
Damaged cells
0
1
2
3
Score
Negative control Positive control DFB 0.3mg/kg (GI) DFB 1.0 mg/kg (GII) DFB 3.0 mg/kg (GIII)
24.20 ± 2.88a 88.40 ± 1.71b 89.40 ± 1.01b 91.50 ± 0.52b 93.50 ± 0.65b
75.80 ± 2.88 11.30 ± 1.80 10.60 ± 1.01 8.40 ± 0.50 6.50 ± 0.65
20.80 ± 2.52 61.10 ± 2.81 66.20 ± 2.27 49.30 ± 1.22 43.20 ± 1.21
3.20 ± 0.55 21.50 ± 2.19 20.00 ± 1.99 34.80 ± 1.64 41.90 ± 1.16
0.20 ± 1.33 5.80 ± 0.78 3.20 ± 0.70 7.40 ± 0.54 8.40 ± 0.83
27.80 ± 3.34a 121.50 ± 4.34b 115.80 ± 2.67b 141.10 ± 2.02c 152.20 ± 2.17c
Note. Data presented as mean ± SEM. Different letters indicate statistically significant differences (p < .05; ANOVA/Tukey). Class 0, no damage; class 1, tail of comet shorter than the diameter of nucleiod; class 2, tail of comet once or twice the diameter of nucleoid; class 3, tail of comet more than twice the diameter of nucleoid.
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TABLE 2. Frequency of Micronuclei Time (h) Treatments
24
48
72
Negative control Positive control DFB 0.3 mg/kg (GI) DFB 1 mg/kg (GII) DFB 3 mg/kg (GIII)
1.50 ± 0.65a 21.50 ± 1.94b 16.80 ± 1.58b 17.60 ± 1.66b 34.10 ± 2.16c
1.00 ± 0.37a 15.00 ± 1.19b 11.40 ± 1.16b 16.30 ± 1.42b 27.90 ± 1.62c
0.90 ± 0.31a 9.70 ± 0.40b 9.80 ± 0.79b 12.40 ± 0.96b 18.90 ± 1.33c
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Note. Data presented are mean ± SEM, frequency of micronuclei per 2000 cells. Different letters indicate statistically significant differences, ANOVA/Tukey (p < .05).
dose (3 mg/kg) showed a significantly higher incidence of MN than the positive control group (cyclophosphamide). Data demonstrate that DFB exerts mutagenic effects in a dosedependent manner. It was found that the frequency of MN decreased chronologically after treatment. This decrease may be due to metabolism of DFB which then reduces its ability to induce DNA damage. Data demonstrated that DFB possesses genotoxic and mutagenic capacity to affect nontarget organisms in a dose-related manner. REFERENCES Agencia Nacional de Vigilância Sanitária. 1985. Índice Monográfico. D17 Diflubenzuron. http://portal.anvisa.gov.br/wps/wcm/ connect/af1db28047458f 8d98c8dc3fbc4c 6735/d17_novo.pdf?MOD=AJPERES&use DefaultText=0&useDefaultDesc=0. De Oliveira, P. R., Bechara, G. H., Denardi, S. E., Oliveira, R. J., and Mathias, M. I. C. 2010. Genotoxic and mutagenic effects of fipronil on mice. Exp. Toxicol. Pathol. 64: 569–777. Fenech, M. 2000. The in vitro micronucleus technique. Mutat. Res. 455: 81–95. Fenech, M., Holland, N., Chang, W. P., Zeiger, E., and Bonassi, S. 1999. The human micronucleus project—An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutat. Res. 428: 271–283. Gontijo, A. M. M. C., and Tice, R. 2003. Teste do cometa para a detecção de dano no
DNA e reparo em células individualizadas. In Mutagênese ambiental, ed. L. R. Ribeiro, E. K. Salvadori, and E. K. Marques, 247–275. Canoas, Brazil: ULBRA. Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., and Ishidate, M., Jr. 1990. The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. Mutat. Res. 245: 245–249. Peirce, J. J., Weiner, R. F., and Vesilind, P. A. 1998. Meteorology and air pollution. In Environmental pollution and control, 271–286. Boston, MA: ButterworthHeinemann. Preston, R. J. 2007. Epigenetic processes and cancer risk assessment. Mutat. Res. 616: 7–10. Reynolds, S. E. 1987. The cuticule, growth regulators and moulting in insects: The essential background to the action of acylurea insecticides. Pestic. Sci. 20: 131–146. Silva, M. T. B., Costa, E. C., and Boss, A. 2003. Control of Anticarsiagemmatalis Hübner (Lepidoptera: Noctuidae) larvae with insect growth regulators. Cienc. Rural. 33: 601–605. United Nations Environment Programme, International Labour Organisation. 1996. Environmental health criteria 184. Diflubenzuron. Geneva, Switzerland: World Health Organization. U.S. Environmental Protection Agency. 1997. Reregistration eligibility decision— Diflubenzuron. Washington, DC: Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA.
